The enzyme ATP/CTP:tRNA nucleotidyltransferase, which catalyzes ligation of CMP or AMP residues onto the 3' end of tRNAs that lack all or part of the universally conserved CCA sequence, is an essential biosynthetic enzyme in eukaryotes and an important repair enzyme in prokaryotes. It is an essential enzyme for rapid growth of cells, and as such is a potential target for inhibiting pathogenic or malignant growth. Understanding how this enzyme achieves its substrate specificity (all tRNAs regardless of their source or amino acid specificity) should provide insight into how proteins recognize secondary and tertiary structural features of RNA. Regions of the protein that mediate specific binding of the tRNA substrate (as well as those involved in catalysis and binding co-substrates ATP and CTP) are not well characterized. The working hypothesis of this research proposal is that a sequence of 11 amino acids, which is highly conserved among eubacterial, archaebacterial, and eukaryotic nucleotidyltransferase genes, is involved in specific recognition of tRNA substrates. Furthermore, a potentially homologous sequence in the HIV-1 rev protein (Rev) is identified in this proposal. The proposed experiments will test the structural and functional homology of Rev and archaebacterial sequences, as well as characterize the putative role of the conserved region in mediating substrate binding. Two experimental approaches will be taken: 1) synthetic peptides, whose sequences match those in cca enzymes and HIV-1 Rev, will be characterized for content of a -helix and as potential inhibitors of substrate binding; and 2) two archaebacterial cca genes will be subcloned and overexpressed. Charged residues within the conserved region will be altered to determine if amino acid substitutions affect substrate recognition. Attempts will also be made to crystallize the overexpressed enzyme for future x-ray diffraction analyses.